Functional Testing of KLF7 in Spinal Cord Injury: An Optogenetic Approach

描述（由申请人提供）：中枢神经系统（CNS）轴突再生的失败阻碍了广泛的中枢神经系统疾病的治疗，包括脊髓损伤，中风和帕金森病等疾病。再生失败的一个主要原因是许多中枢神经系统在轴突生长的内在能力方面经历了发育损失。为了恢复中枢神经系统损伤或疾病后的功能，我们必须设法提高中枢神经系统神经元的内在生长状态。我们最近发现转录因子家族（KLFs）是中枢神经系统神经元内在再生能力的关键调节因子。关键是，转录活性突变VP16-KLF7促进受损皮质脊髓束（CST）神经元的轴突再生，这是一个重要的治疗靶点。通过将病毒传递给成年野生型动物，实现了强大的再生反应，提高了潜在的临床

DESCRIPTION (provided by applicant): The failure of axon regeneration in the central nervous system (CNS) prevents treatment of a wide range of CNS afflictions, including spinal cord injury, stroke, and diseases like Parkinson's. One major reason that regeneration fails is that many CNS undergo a developmental loss in their intrinsic capacity for axon growth. To restore function after CNS injury or disease it is essential that we devise means to enhance the intrinsic growth state of CNS neurons. We recently identified the Kr¿ppel-like family of transcription factors (KLFs) as key regulators of intrinsic regenerative capacity in CNS neurons. Critically, a transcriptionally active mutant, VP16-KLF7, promotes axon regeneration by injured corticospinal tract (CST) neurons, an important therapeutic target. Robust regenerative responses were achieved using viral delivery to adult wild-type animals, raising potential clinical relevance for this novel pro-regenerative tool. To further explore the potential of KLF7 activity t promote CNS regeneration, three key questions must be addressed, corresponding to our aims. First because recovery from SCI depends on multiple functional modalities carried by diverse fiber tracts, we will use viral-mediated gene delivery to test the ability of VP16-KLF7 to promote regeneration in additional neuronal populations. Targeting additional neuronal subtypes has the potential to broaden the range of behavioral improvement after SCI. Second, we will examine the expression of pro-regenerative genes in neurons stimulated by VP16-KLF7 in order to determine the potential relationship between with known pro-regenerative (e.g. mTOR, CNTF) or anti-regenerative (e.g. chondroitin sulfate proteoglycan (CSPG)) pathways and signals. Based on this information we will rationally combine VP16/KLF7 with viral particles that target complementary pathways, potentially inducing additive or synergistic improvements in axon regeneration. Finally, we will examine the extent to which KLF7-stimulated axons succeed in forming functional synapses on appropriate target cells and ultimately contribute to functional recovery. Using viral gene delivery we will co-express VP16- KLF7 with optogenetic constructs that enable the treated neurons to be reversibly activated or silenced. This technique will allow us to determine the specific contribution of the treated neurons to electrophysiological and behavioral output as regeneration proceeds. Taken together, these experiments have a strong potential to extend the use of a promising new pro-regenerative tool and lead to the development of novel combinatorial methods to promote axon regeneration and functional recovery in the injured CNS, leading to novel treatment options for people suffering from CNS afflictions.